Re-examination of the snow algal species Chloromonas miwae (Fukushima) Muramoto et al., comb. nov (Volvocales, Chlorophyceae) from Japan, based on molecular phylogeny and cultured material

Carteria (Ca.) miwae was identified as a volvocalean snow alga distributed in Japan. Although Ca. miwae was recorded from various mountainous areas of Japan in its original description, this species has not been studied in over 40 years. We re-examined Ca. miwae, using culture techniques, electron microscopy and molecular phylogenetic analysis on snow samples collected from Mt. Hakkoda, Aomori Prefecture, and Mt. Gassan, Yamagata Prefecture, Japan. Light and transmission electron microscopy (TEM) of ellipsoidal or elongate-ovoid quadriflagellate cells from field samples revealed the presence of two eyespots and the absence of typical pyrenoids surrounded by starch grains in the chloroplasts, which are both characteristic of Ca. miwae. Single-cell sequencing demonstrated that quadriflagellate and naked biflagellate cells with a single eyespot growing in the same snow sample had identical gene sequences (1128 bp) in the large subunit of Rubisco (rbcL), suggesting that these two cell types represent different stages in the sexual cycle of a single species. Our molecular phylogenetic analysis showed that the quadriflagellate and biflagellate cells, and two new strains of the biflagellate volvocalean genus Chloromonas (Cr.) in the Japanese snow samples, form a small clade within the Chloromonas snow taxa. Thus, quadriflagellate cells can be considered planozygotes within the life cycle of a Chloromonas species growing in snow in Japan, and the new combination Chloromonas miwae (Fukushima) Muramoto et al. is proposed, based on Carteria miwae. The unique morphological characteristics and distinct phylogenetic position of Cr. miwae revealed in the present study suggest that this Japanese species has its own peculiar evolutionary history within the worldwide Chloromonas snow taxa.